Figure 4

Distinct phenotypes of subpopulation cells in U251.

A, real-time qRT-PCR (right) quantification of the expression of genes associated with neural stem cell features as well as glioma cell migration and invasion, normalized to ACTB, in cells of the same cultures used for zymography, and enzyme immunometric assays (left) for quantification of VEGFA (VEGF-165) and SPP1 (Osteopontin) in conditioned medium, normalized by cell numbers. Bar and line height are mean and SD based on quantification of 3–6 sets of independent cultures. B, western blot (top) and immunocytofluorescence (bottom) of EGFR (1∶1000 from Cell Signaling) in U251 parental (P) and two clonal NS lines (NS1 and NS2). C, soft agar colony formation assay of U251 parental (P), NS1, and P-E1wd lines. D, s.c. tumorigenicity assay of cells described above, with follow-up of tumor growth as described previously [33]. E, immunocytofluorescence analysis of NS1 and SA1-NS before and after being subjected to neural stem cell differentiation conditions described in Methods.